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Salinity stress effects on three different olive cultivars and the possibility of
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Plant Archives Vol. 20, Supplement 2, 2020 pp. 2378-2382 e-ISSN:2581-6063 (online), ISSN:0972-5210

SALINITY STRESS EFFECTS ON THREE DIFFERENT OLIVE CULTIVARS AND THE

POSSIBILITY OF THEIR CULTIVATION IN RECLAIMED LANDS
1 1 1 2 2
Islam F. Hassan * , Gaballah Maybelle , Abou Leila Bedour , Primo Proietti and Luca Regni
1
Water Relations and Field Irrigation Department. Agriculture and Biological Research Division, National Research Centre,
Cairo, Egypt.
2
Department of Agricultural, Food and Environmental Sciences, University of Perugia, Perugia, Italy.
Corresponding Author: islam_frahat@yahoo.com

Abstract

The use of underground saline water for irrigation became essential in agricultural lands of many countries .Twelve-month-old olive young
trees (Olea europaea L. cv Picual, Manzanillo and Aggizi Shami) were exposed to different salinity levels (0, 2000, 3000 and 4000 mg L-1)
for 275 days in order to understand the differences among those cultivars, regarding salinity resistance. In specific, the main aim was to
select the suitable cultivar for growing in the reclaimed lands in Egypt, which are suffering from poor irrigation sources, forcing the growers
to use underground saline water. At the end of this experiment the effects of different salinity concentrations (0, 2000, 3000, 4000 mg L-1),
on growth, physiological and biochemical parameters were determined. The obtained results assumed that, the tolerance to salinity stress
was as follows: Picual > Manzanillo > Aggizi Shami. The previous order of salt stress tolerance was proven by a lower decrement in
vegetative growth parameters (seedlings height, number of leaves and leaf area), relative water content, total chlorophyll content, and greater
increment of proline, soluble carbohydrates, and electrolyte leakage. Also the capacity of the elimination procedure of Na+ and Cl_ in the
root system.
Keywords: Olea europaea; salt stress; reclaimed land; plant growth

Introduction
Salinity of water is one of the main abiotic stresses that
reduce plant improvement and crop production in the world.
Salinity stress in most cases decreases vegetative growth and
biochemical constitution [Bose and Shabala, 2015; Pandolfi
et al., 2017; Hassan I. F et al., 2019]. In the Mediterranean
region, especially in the arid regions where olive is mostly
grown, there is an increasing request for the water of
excellent quality. Hence, the availability of water for
irrigation is declining in these regions [Chartzoulakis, 2005].
To supplement the lack of water and satisfy the increasing
need for agricultural growth, the use of underground saline
water for irrigation is becoming essential in many countries.
Very little work is available on the tolerance of Picual and
Manzanello under the Egyptian conditions and no work has
been conducted on the tolerance of Egyptian olive cultivar
Aggizi Shami to salinity stress [Chartzoulakis, 2005;
Chartzoulakis et al ., 2002 ; Perica et al., 2008 ]. In this
situation, it is vital to choose suitable cultivars, which will
grow well when planted in the newly reclaimed lands
irrigated with underground saline water. Most of the
Egyptian olive farmers use local varieties of olive, with very
few them growing the newly imported varieties of olive,
without recognizing their suitability to the Egyptian
conditions in general and the high salinity in particular.
Hence, we selected three different cultivars namely Picual,
Manzanillo and Egyptian Aggizi Shami to compare and
select the most suitable cultivar for growing in our reclaimed
lands at El Sadat City, Egypt. In most of the cases, water
used for irrigation at the reclaimed lands in El Sadat city is
saline groundwater. Furthermore, the cultivars grown at these
lands are generally imported cultivars. Picual and
Manzanillo. Salinity resistance in olive, as in other species, is
cultivar-dependent [Chartzoulakis et al., 2002; Perica et al.,
2008], there are few studies that compare olive cultivars of
different origins to assess the differences among them [Perica
et al., 2008]. Olive trees under high salinity conditions are
subject to physiological and biochemical alterations, for
example, a marked decrease in photosynthesis rate that leads
to a reduction in plant growth (mainly shoot growth). Salinity
can straightly affect the absorption of nutrient ions
absorption, sodium and chloride reduce potassium and nitrate
absorption, respectively [Ben Ahmed et al., 2009; Regni et
al., 2019; Mousavi et al., 2019]. This study was conducted to
explore salinity tolerance of Picual, Manzanillo against the
local variety Aggizi Shami to determine which variety is well
adapted to grow under saline irrigation conditions of the
Egyptian reclaimed lands.
Material and Method
Plant material, growing conditions and salt treatments
The experiment was conducted from 15th February to
15th November 2019 (275 days) on three different olive
cultivars (Picual, Manzanillo and Aggizi Shami). One-year-
old own-rooted young seedlings similar in trunk diameter and
height were selected, and transferred to grow in a slightly
shaded and unheated greenhouse located in a private farm at
El Sadat City, Egypt. The young seedlings were cultivated in
pots of 2 kg capacity in a mixture of sand: clay in 2:1 ratio,
each pot had one plant. The young seedlings were kept one
month for adaptation to the new growing conditions before
starting the experiment. Young olive seedlings were irrigated
two times per week by different concentrations (control,
freshwater, 2000, 3000 and 4000 mg L-1) of saline solution
as shown in table 1, consisted of a mixture of salts according
to [Stroganov, 1962] and one time by freshwater to avoid
osmotic shock. Every pot watered until the volumetric
 Islam F. Hassan et al. 2379

moisture content which correspond to the field capacity the
voultmtric. All the agricultural fertilization programs for
olive seedlings, recommended by the Egyptian ministry of
agriculture were used.
Table 1. The content of salt solution used for salinization
expressed as % of total salt content.
MgSO4 CaSO4 NaCl MgCl2 CaCO3
% % % % % RWC (%) = [(FW-DW) / (TW-DW)] x 100 (2)
10 1 78 2 9
Plant height, leaf number and leaf area
At the end of the experiment the length of the principal axis
(cm) was measured as well as the number of leaves per plant. In
addition, leaf area (cm2) was determined by the equation described
by [Singh and Snyder, 1984].
Total chlorophyll, proline, soluble carbohydrate, Na+ and Cl-
contents
At the end of the experiment, the leaf total chlorophyll content
was measured according to [Saini, 2001], by extracting in 85%
acetone solution and measuring their absorbance by using a
Spectrophotometer at λ= 663 nm and 645 nm. The amount of total
chlorophyll was calculated using the following equation:
Total chlorophyll (mg /g fw) = [(20.2×OD 645 nm + 8.02×OD 663
nm) ×V] /(fw ×1000) (1)
OD is optical density, V is the final solution volume in mL
and fw is tissue fresh weight in mg. V is the final solution volume in
mL and fw is tissue fresh weight in mg.
Leaf proline was extracted from the 0.5 g samples of young
leaves by 3% sulfuric acid and determined by using the ninhydrin
reagent, according to the method described by [Bates et al., 1973].
The absorbance of the solution with toluene was determined at 520
nm, using a spectrophotometer (Model UV-120-20, Japan)
percentages according to the method described in [Chapman and
Pratt,1961].
Leaf relative water content and electrolyte leakage
The leaf relative water content was estimated according to the
method described by [Saini, 2001], performed by incubating 0.2 g
of fresh leaf sample in 50 mL of distilled water for 4 h. Then the
turgid weights of leaf samples were measured. The leaf samples
were oven-dried at 60°C for 48h for dry weight determination. The
RWC was calculated using the following equation,
Where FW, DW, and TW are fresh, dry and turgid weights.
For the electrolyte leakage ten discs of fresh expanded leaves
(0.5 cm diameter) were cut from the fully expanded leaves
according to the method of [Saini, 2001]. The leaf discs were
washed three times with deionized water to remove dust and put in
closed tubes containing 10 ml of deionized water and shake for 30
minutes and left in dark at room temperature for 24 h. The initial
electrical conductivity of the solution (EC1) was determined using
an electrical conductivity meter. The samples were put in a water
bath at 80°C for 20 min to release all the endogenous electrolytes
and cooled to 25 °C,. Finally, their electrical conductivity (EC2)
was measured.
EL = (EC1/EC2) × 100 (%). (3)
The solution discharge (EL) was calculated per the subsequent
equation:
Experimental design and statistical analysis
The experimental design was split plot design including three
cultivars and four salinity concentrations and six replicates. The
obtained data were subjected to variance analysis according to
[Clarke, and Kempson, 1997]. The means were differentiated using
the Rang test at the 0.05 level [Duncan, 1995].
Results
Plant height, leaf number and area, leaf total chlorophyll
content

The soluble carbohydrate concentration was determined in
dried leaf samples according to [Buysse and Merckx, 1993], by
extracting 150 mg of dried leaf samples twice with 80% ethanol.
The sample was centrifuged at 3500 rpm for 10 min and the volume
of the supernatant was adjusted to 25 ml. Then 1ml of supernatant
was transferred to a test tube and 1 ml phenol (18%) and 5 ml
sulfuric acid were added. The mixture was shaken immediately and
its absorption was recorded at 490 nm using a spectrophotometer
(Model UV-120-20, Japan).
The data presented in Table 3 indicated that, all salinity
treatments had a harmful effect on the cultivars vegetative growth
(plant length, leaf area, and number of leaves). The effect of salinity
on theses measurements showed significant genotypic differences
associated with higher concentration of salinity from 0 to 4000 mg
L-1 compared with the control plants. The Picual cultivar showed
higher values of plant height, number of leaves and leave area
compared to Manzanillo and Aggizi Shami as follows. The
differences between cultivars were significant.

The shoots and roots were collected at the end of the

experiment in order to determine the sodium and chloride

Table 2. Effect of saline water concentrations on seedlings length (cm), Leaf area (cm) and number of leaves of the three olive
cultivars
Characters Seedlings length (cm2) Leaf area (cm2) Number of leaves
Treatments Cultivars Cultivars Cultivars
Salinity (B) Picual Manzanillo Aggizi Shami Mean Picual Manzanillo Aggizi Shami Mean Picual Manzanillo Aggizi Shami Mean
0 mg L-1 84.33a 80.00bc 76.67d 80.33A 4.10b 4.40a 4.03b-d 4.18A 73.67a 72.00b 71.33bc 72.33A
2000 mg L-1 82.00ab 75.33de 70.67f 76.00B 3.97b-e 4.07bc 3.93c-e 3.99B 70.67c 65.00e 64.00ef 66.56B
3000 mg L-1 79.33c 73.67e 66.33g 73.11C 3.90de 3.90de 3.80e 3.87C 66.67d 62.33g 62.00g 63.67C
4000 mg L-1 75.00c 67.00g 60.00h 67.33D 3.87de 3.80e 3.60f 3.76D 63.67f 60.33h 56.33i 60.11D
Mean 80.17A 74.00B 68.42C 2085B 4.04A 3.84C 68.67A 64.92B 63.42C
Means marked by the same letter are not significantly different at p = 5% level, using Duncan’s test.
 Salinity Stress Effects on Three Different Olive Cultivars and The Possibility of Their Cultivation in Reclaimed Lands 2380

Table 3. Effect of saline water concentrations on total chlorophyll mg g-1 fw , proline µg g-1fw and Soluble sugars mg 100g-1
dw content
Total Chlorophyll mg g-1 fw Proline µg g-1 fw Soluble sugars mg 100g-1 dw
Treatments Cultivars Cultivars Cultivars
Salinity (B) Picual Manzanillo Aggizi Shami Mean Picual Manzanillo Aggizi Shami Mean Picual Manzanillo Aggizi Shami Mean
0 mg L-1 2.45a 2.39a 2.21b 2.35A 24.00d 22.00e 25.33d 23.78D 26.67g 32.00f 31.00f 29.89D
2000 mg L-1 2.19bc 2.03d 2.08cd 2.10B 27.33c 25.00d 27.33c 26.56C 25.67g 36.00de 37.00cd 32.89C
3000 mg L-1 1.98de 2.01de 1.82f 1.94C 31.67b 28.67c 34.33a 31.56B 35.00e 39.67b 38.00c 37.56B
4000 mg L-1 1.89ef 1.80f 1.80f 1.83D 31.00b 32.67b 35.00a 32.89A 38.33bc 39.67b 43.00a 40.33A
Mean 2.13A 2.06B 1.98C 28.50B 27.08C 30.50A 31.42B 36.83A 37.25A
Means marked by the same letter are not significantly different at p = 5% level, using Duncan’s test.

Table 4. Effects of saline water concentrations on relative water content and electrolyte leakage.
Characters Relative water content % Electrolyte leakage %
Treatments Cultivars Cultivars
Salinity (B) Picual Manzanillo Aggizi Shami Mean Picual Manzanillo Aggizi Shami Mean
0 mg L-1 91.00a 84.67b 83.67b 86.44A 10.33j 17.00i 16.33i 14.56D
2000 mg L-1 81.67c 80.00d 81.33c 81.00B 19.67h 23.67f 21.33g 21.56C
3000 mg L-1 80.67cd 75.00f 71.00h 75.56C 21.00g 25.00e 35.00b 27.00B
4000 mg L-1 78.67e 73.33g 69.00i 73.67D 31.67c 26.67d 36.67a 31.67A
Mean 83.00A 78.25B 76.25C 20.67C 23.08B 27.33A
Means marked by the same letter are not significantly different at p = 5% level, using Duncan’s test.

Na+, Cl- and contents in both roots and leaves

Total chlorophyll, proline and soluble carbohydrate contents
The data presented in Figures 1 and 2 illustrated that, both
The obtained results presented in Table 4, demonstrated
sodium and chloride concentrations in the different seedlings
that all the biochemical constitutes of the tested olive cultivars
parts (roots and leaves) increased gradually by the increase in
were significantly affected by the increment of water salinity
salinity concentrations from 0 to 4000 mg L-1. A significant
concentrations. The total chlorophyll decreased but soluble
variation was recorded between the leaves and roots in sodium
sugars and proline contents increased by increasing salinity
content compared with the control seedlings. Picual cultivar
concentrations compared to control seedlings. The Picual
roots accumulated higher sodium content than leaves while
cultivar recorded higher values of total chlorophyll content than
Aggizi Shami leaves recorded a higher Na+ content than roots.
Manzanillo and Aggizi Shami, while Aggizi Shami recorded the
As for chloride, concentrations both (in leaves and in roots)
higher values of proline content and soluble sugars followed by
increased gradually with salinity concentration ranging within 0
the Picual and Manzanillo. Regarding, the interaction between
to 4000 ppm. Chloride concentrations were higher in roots than
salinity and cultivars, Picual cultivar recorded higher total
in leaves compared with the control seedlings. A remarkable
chlorophyll content when irrigated with 2000 mg L-1 and lower
variation was noticed among cultivars, Picual cultivar
proline and soluble sugars contents, while the Aggizi Shami
accumulated higher chloride content in roots , while Aggizi
cultivar recorded higher proline and soluble sugars when
Shami cultivar accumulated lower chloride content and
irrigated with 4000 mg L-1 and lower total chlorophyll content
Manzanillo cultivar ranked in between.
than control plants .

Water status: leaf relative water content (%) and electrolyte

leakage (%)
The results in Table 5 demonstrated that increasing salinity
concentrations gradually decreased, the leaf relative water
content in all cultivars compared with the control, while the
electrolyte leakage increased by increasing salinity
concentrations from 0 to 4000 mg L-1. Leaf water content
showed high significant differences among the studied cultivars.
Picual cultivar showed higher relative water content (83.00%),
while Aggizi Shami (76.25%) showed the lower relative water
content Manzanillo showed an intermediate value (78.25%) of
relative water content.Ectrolyte leakage percentage varied
among the cultivars; the results showed that higher value of
electrolyte leakage percentage was recorded by Aggizi Shami
(27.33%) and the lower value was recorded by Picual cultivars
(20.67%). Concerning the interaction between salinity and
cultivars, Picual cultivar recorded higher relative water content
when irrigated with 2000 mg L-1 and lower electrolyte leakage
percentage, while Aggizi Shami cultivar recorded higher
electrolyte leakage percentage when irrigated by 4000 mg L-1
and lower relative water content. Manzanillo ranked in between. Figure 1. Effects of saline water concentrations on sodium
percentage content in both roots and leaves.
 Islam F. Hassan et al.
Figure 2. Effects of saline water concentrations on chloride
percentage content in both roots and leaves.
Discussion
The results of present research showed a reduction in the
vegetative growth parameters namely young tree height, leaf area
and number of leaves. In all the three studied cultivars by the
increasing of salinity concentrations from 2000 mg L-1 to 4000 mg,
L-1 compared to the control plants. The vegetative growth reduction
and onset of destruction are further correlated with the Na+
accumulation in leaves that leads to salinity toxicity causing leaf
drop [Therios and Misopolinos,1988; Tattini et al., 1992]. Many
previous studies reported that shoot growth is more sensitive to salt
stress than root growth [ Moran et al ., 1994 ;Tattini et al ., 1995;
Chartzoulakis et al., 2002]. When using these vegetative growth
parameters as the indicators of resistance, Picual can be considered
as the most tolerant cultivar to salinity, followed by Manzanillo and
Aggizi Shami.
A remarkable salt tolerance mechanism was found in Picual
cultivar, maybe being associated with the salt accumulation process
at the root level, which reduced Na+ accumulation in leaf tissues
described by [Therios and Misopolinos,1988; Chartzoulakis].
Indeed, Na+ and Cl- ions concentrations in Picual leaves were lower
than the other cultivars while their concentration in the root was
higher than the other cultivars. In line with our data, [Chartzoulakis
et al., 2002] revealed that salinity resistance in olive cultivars
interacted with the efficient performances of ion insularity and
limited sodium and chloride concentrations in the roots. Our data
proved the occurrence of vital differences between the studied
cultivars concerning the expansion and transport of sodium and
chloride in roots and leaves.
In our experiment, the total chlorophyll content decreased
among all the studied cultivars by the increase of salinity
concentrations, which may be attributed to various reasons such as
the inhibition of chlorophyll biosynthesis by the activation of
chlorophyllase deterioration, resulted from salinity-mediated
chlorophyll degradation. [Younis et al., 2000; Yasar et al., 2008;
Gill and Tuteja, 2010]. Also, the salinity increases ROS which
causes the osmotic and hormonal imbalances in plant cells [Gill and
Tuteja, 2010; Saha et al., 2010, Din et al., 2011 ; Arjenaki et al.,
2012, Feller and Vaseva, 2014]. The accumulation of osmotic
protectants plays a vital role in maintaining the intracellular stability
and saving cells from the harmful effects of salt and toxicity.
The proline and soluble sugars concentrations in the three
tested cultivars under salinity conditions varied, higher in
Manzanillo and Aggizi Shami and less in Picual compare with
control trees. In saline-stressed trees, proline and soluble sugars
2381
increments were statistically significant between the three cultivars,
these results in harmony with [Parida and Das, 2005; Munns and
Tester, 2008 Hayat et al., 2012; Regni et al., 2019]. In this regard, it
must be noted that they correlate the trend affecting proline and
soluble sugars concentrations with the ability to exclude Na+ and Cl
from roots to leaves between the different cultivars. The increase in
osmolytes as proline or soluble sugar derived from the metabolisms
of protein and carbohydrates is an adaption tool of the plant to
reduce the harmful effect of salinity [Parida and Das, 2005; Iqbal et
al., 2014; Regni et al., 2019]. Definitely, proline increases the water
retention in the cytoplasm and its higher content appears to be a
specific mechanism involved by the plants to further tolerate
moderate stress conditions [Parida and Das, 2005; Munns and
Tester, 2008; Ben Ahmed et al ., 2009; Hayat et al., 2012]
The relative water content declined in all the cultivars exposed
to salt stress and the main devaluation was noted in Egizzi Shamii
cultivar. The relative water content and electrolyte leakage are
important physiological parameters for measuring the water status
of the plants. As found in our results the RWC decreased in all the
studied cultivars by increasing the salinity from 0 to 4000 ppm,
these results were in harmony with [Perica et al., 2008]In general,
the salinity stress affected the plant water relations, whereas RWC
decrease resulted from high salinity concentrations in the external
soil solution caused osmotic stress leading to dehydration and
increase in electrolyte leakage values. The increase in electrolyte
leakage might be because of the accumulation of ROS because of
salinity in plant cells, which induced cell membrane injury [Lutts et
al ., 1996 Dionisio-Sese and Tobita ;Cardi , 1998 ; Franklin and
Zwiazek, 2004 ; Goreta et al ., 2007 ; Cardi et al ., 2015]The novelty
of our research is the long period (275 days) of our experiment
whereas most of the former studies have been conducted for short
period ranged between 150 and 240 days [Chartzoulakis et al ., 2002
; Ben Ahmed et al ., 2009; Regni et al., 2019]. However, it could be
figured that the difference between the studied cultivars grown in
pots may be due to the duration of salinity treatment, which was
enough to show the differences under controlled conditions (275
days), to determine which variety is suitable to grow under
reclaimed lands conditions. Our results showed that cv Picual olive
trees are well suited to reclaimed lands in Egypt and the most
tolerant among the studied cultivars to increased salinity conditions
from 2000 to 4000 ppm maintaining the greater photosynthetic
activity and vegetative growth. However, no toxicity effects were
seen at all concentrations. The increments of proline and soluble
sugars accumulation under salinity conditions indicated a direct
relationship of this osmolyte with salinity tolerance of the Picual
olive cultivar in performance with the dehydration prevention
mechanisms such as stomata closure to reduce water loss .
Conclusion
In conclusion, our results cleared showed a different response
to salinity by the tested cultivars. Picual was the most tolerant,
showing a lower reduction of growth and ability to limit the increase
of leaf Na+ concentration. The growth of ‘Aggizi Shami’ was
highly suppressed by salinity, and together with ‘Manzanillo’ it
showed the higher Na+ accumulation in leaves. The obtained results
suggested that Picual cultivar might be the suitable cultivar to be
grown in reclaimed lands irrigated by the underground saline water.
Funding: This work was partially funded by National Research
Center, Cairo, Egypt and university of Perugia, Italy.
Acknowledgment: We would like to thank greenhouse technical
workers Mr. Sayed and Mr. Ibrahim for helping during the
experimental work and the laboratory technical staffs Mr.Hussein
and Mr. Hamdy for helping during the laboratory work.
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